Angiogenesis, the formation of new blood vessels by endothelial cells, plays a critical role in wound healing, the revascularization of ischemic organs, and the progression of atherosclerosis and cancer. The basic theme of this grant is that periendothelial mural cells regulate angiogenesis by paracrine mechanisms. Our previous work focused on the role of the mural cell-derived product angiopoietin-1 (Ang-1) and its receptor Tie2 in the aortic ring model of injury-induced angiogenesis. The following novel findings emerged from these studies. 1. Ang-1 induces expression of tumor necrosis factor-alpha (TNF) and other immune-related cytokines/chemokines, which stimulate macrophage recruitment and angiogenesis. 2. TNF is required for the angiogenic activity of Ang-1-induced cytokines. 3. Ang-1-mediated mural cell recruitment can be inhibited by blocking the p38 MARK pathway, which regulates production of monocyte chemotactic protein-1 (MCP-1), a mural cell stimulatory chemokine induced by Ang-1. 4. Ang-1 promotes expression of Toll-like receptor-2 (TLR2) and the TLR4 adapter molecule CD14, which regulate cytokine production in the innate immune system. 5. Stimulation of TLR2 with specific ligands dose-dependently promotes angiogenesis. 6. The angiogenic response of the aortic wall is impaired in mice with disrupted MyD88, a key TLR signaling molecule. On this basis we formulated the hypothesis that Ang-1 regulates angiogenesis in response to injury by cooperating with the innate immune system. Specific aims: (1) To establish the role of vascular macrophages and their cytokine product TNF in Ang-1-mediated angiogenesis. (2) To define the role of MCP-1 in Ang-1/p38 MAPK-mediated mural cell recruitment during angiogenesis. (3) To determine the function of TLRs in the angiogenic response of the vessel wall. For these studies we will use rat and mouse in vitro and in vivo models of angiogenesis. Functional studies will be performed with neutralizing antibodies and genetically modified mice with defective TNF and TLR systems. Macrophages will be selectively ablated using biochemical and transgenic methods. Molecular studies will include real-time RT-PCR, ELISA, laser capture microscopy, and confocal microscopy. Proposed experiments will define new molecular mechanisms of angiogenesis and vessel wall assembly. This knowledge may provide novel insights for the development of therapeutic approaches aimed at treating angiogenesis-dependent disorders and ischemic conditions.